The relationships of the snRNA structural elements to their functions in spliceosome assembly and splicing are only generally understood. A persistent mystery in splicing is how the many RNA helicase family members achieve snRNA rearrangements during assembly of the spliceosome, catalysis, and disassembly. A second major question concerns how splicing regulators influence the basic process of spliceosome assembly. Many proteins that regulate splicing are known, but exactly how they do it remains quite opaque. A third and emerging set of questions concerns the role of splicing and other small RNA-mediated processes in genome function and evolution. We will address each of these major questions with a specific aim. (1) We will dissect functional interactions between the DEAD-box RNA helicase family member PrpSp and components of the U2 snRNP in particular Cus2p and U2 snRNA during spliceosome assembly and splicing in vitro and in vivo. Cus2p and PrpSp influence U2 snRNA structure and each other to control the delivery of U2 snRNA to the branchpoint region of the pre-mRNA in an ATP-dependent step-prespliceosome assembly. (2) We will explore the mechanism of Mer1p-activated splicing and its importance for the meiotic gene expression program in yeast. Since Merlp binds U1 snRNPs, we will focus on the composition and activities of the U1 snRNP with and without Mer1p. A second part of this aim is to understand the roles of Mer1p and the four known genes whose splicing depends on Mer1 p in the meiotic gene expression program using splicing sensitive microarrays. (3) We will study the integration of splicing with other steps in the gene expression pathway and will begin to determine how the loss of introns containing conserved sequence affects cellular function. A large microarray study completed in the last funding period presents several specific hypotheses for how splicing and small RNAs are connected to other steps in gene expression. The areas of inquiry represented by these aims encompass the major issues in the splicing field: How do snRNPs work? How is splicing regulation achieved? And how is the process of splicing integrated into genome function and evolution? The combined strengths of genetics, biochemistry, and genomics available in yeast and the conserved properties of the splicing machinery indicate that fundamental knowledge uncovered by these efforts will translate directly to the understanding of human gene expression.